Mechanism Of Cadmium-induced Inflammation And Autophagy In Common Carp(Cyprinus Carpio L.) Hearts

Cadmium(Cd)is a toxic heavy metal in the environment.With industrial activities such as mining and smelting,Cd enters the environment and pollutes the soil and water environment,and then affects the health of animals and human beings.Common carp is a kind of economic animal which is raised in great quantity.Heart is an important organ in vertebrate circulatory system.It has been shown that Cd in the accumulation of organism affects the normal function of the heart and has toxic effect on the heart,but the molecular mechanism of Cd-induced cardiac injury in common carp is still unclear.Therefore,in this study,common carp as the research object,successfully established Cd poisoning model,and randomly divided fifty-four juvenile common carp into two groups:control group and Cd group.In the Cd group,the Cd concentration in the water was 0.26mg/L(1/25 of 96h LC₅₀ in common carp).Heart samples were collected on the 45^th day of Cd exposure in common carp.The changes of microstructure and ultrastructure were observed by optical microscope and electron microscope respectively in common carp hearts.The activities of AST,LDH and?-HBDH in serum were detected by biochemical analyzer.According to the instructions,the kits were used to detect CK activity,eight oxidative stress-related indicators(H₂O₂,MDA,GSH,CAT,SOD,GSH-PX,GST,and T-AOC),four ATPases(Na⁺K⁺-ATPase,Ca²⁺-ATPase,Mg²⁺-ATPase and Ca²⁺Mg²⁺-ATPase)activities,and inflammatory-related factors(NO content and i NOS activity).The levels of two heat shock protein-related genes(Hsp70 and Hsp90),five mitochondrial dynamics-related genes(OPA1,Mfn1,Mfn2,Drp1 and Mff),eight inflammation-related factors(NF-?B,TNF-?,IL-1?,IL-6,IL-10,TGF-?,COX-2 and PTGEs),miR-25-3p,miR-9-5p and twelve autophagy-related genes(AMPK,ULK1,PTEN,Sirt1,m TOR,P62,Beclin 1,Dynein,LC3-I,LC3-II,Atg5 and Atg12)were detected by q RT-PCR in order to explore the complex mechanism of Cd exposure to common carp cardiotoxicity.The results showed that:(1)Cd poisoning affected the growth and development of common carp.(2)Cd poisoning led to mitochondrial structure damage,inflammatory cell infiltration and autophagosomes,which proved that Cd poisoning could led to mitochondrial damage,inflammatory injury and autophagy in common carp hearts.(3)Cd poisoning up-regulated the activities of serum cardiac enzymes,which proved that Cd poisoning caused heart damage in common carp.(4)Cd poisoning down-regulated the activities of Na⁺K⁺-ATPase,Ca²⁺-ATPase,Mg²⁺-ATPase and Ca²⁺Mg²⁺-ATPase,decreased the m RNA levels of mitochondrial fusion factors OPA1,Mfn1 and Mfn2,and increased the m RNA levels of mitochondrial division factors Drp1 and Mff,which indicated that mitochondrial dysfunction occurred in common carp hearts.(5)Cd poisoning increased the contents of H₂O₂ and MDA,decreased the contents of GSH and the activities of CAT,SOD,GSH-PX,GST and T-AOC,which indicated that Cd poisoning caused oxidative stress in common carp hearts.(6)Cd poisoning up-regulated the m RNA levels of Hsp70 and Hsp90,which indicated that Hsp was involved in the mechanism of Cd poisoning.(7)Cd poisoning increased the content of NO and the activity of i NOS,increased the m RNA levels of pro-inflammatory factors NF-?B,TNF-?,IL-1?,IL-6,COX-2 and PTGEs,and decreased the m RNA levels of anti-inflammatory factors IL-10 and TGF-?,which indicated that Cd poisoning induced inflammation in common carp hearts.(8)Cd poisoning down-regulated the m RNA levels of m TOR and P62,and up-regulated the m RNA levels of AMPK,ULK1,PTEN,Beclin 1,Dynein,LC3-I,LC3-II,Sirt1,Atg5 and Atg12,which confirmed that Cd exposure induced autophagy in common carp hearts through AMPK/m TOR/ULK1 signaling pathway and Sirt1/m TOR signaling pathway.(9)Through the detection and analysis of miR-25-3p,AMPK,ULK1 and PTEN,this study confirmed for the first time that AMPK,ULK1 and PTEN are the target genes of miR-25-3p,and miR-25-3p mediates Cd induced autophagy.(10)Through the detection and analysis of miR-9-5p and Sirt1,this study confirmed for the first time that Sirt1 is the target gene of miR-9-5p,and miR-9-5p mediates Cd induced autophagy.In conclusion,miRNA,oxidative stress,heat shock protein and mitochondria play important roles in Cd-induced hearts inflammation and autophagy in common carp.